Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein
Abstract
:1. Introduction
2. Results
2.1. Three Min Interval before In Vivo EP Is Effective to Induce Indels in Early Zygotes
2.2. Mode of Distribution of a Solution Injected into the Oviductal Lumen
2.3. Visualization of i-GONAD-Mediated Delivery of Fluorescent Reagents to Early Zygotes
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Mating Protocol
4.3. Preparation of Solutions Used for i-GONAD
4.4. i-GONAD Procedure and Post-i-GONAD Treatment
4.5. Analysis of CRISPR/Cas9-Induced Indels
4.6. Fluorescence Analysis
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CRISPR/Cas9 | clustered regularly interspaced short palindromic repeat-associated protein 9 |
ET | egg transfer |
Fgf10 | fibroblast growth factor 10 |
GM | genetically modified |
i-GONAD | improved genome editing via oviductal nucleic acid delivery |
Indel | insertion and deletion mutation |
HA | hyaluronidase |
Ho33342 | Hoechst33342 |
KI | knock-in |
KO | knock-out |
Pa.m. | protospacer adjacent motif |
PCR | Polymerase chain reaction |
RFD | tetramethylrhodamine-labeled dextran 3 kDa |
tracrRNA | trans-activating cr RNA |
Tyr | tyrosinase |
References
- Wang, H.; Yang, H.; Shivalila, C.S.; Dawlaty, M.M.; Cheng, A.W.; Zhang, F.; Jaenisch, R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013, 153, 910–918. [Google Scholar] [CrossRef] [PubMed]
- Yang, H.; Wang, H.; Shivalila, C.S.; Cheng, A.W.; Shi, L.; Jaenisch, R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 2013, 154, 1370–1379. [Google Scholar] [CrossRef] [PubMed]
- Mashiko, D.; Fujihara, Y.; Satouh, Y.; Miyata, H.; Isotani, A.; Ikawa, M. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci. Rep. 2013, 3, 3355. [Google Scholar] [CrossRef] [PubMed]
- Horii, T.; Arai, Y.; Yamazaki, M.; Morita, S.; Kimura, M.; Itoh, M.; Abe, Y.; Hatada, I.I. Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci. Rep. 2014, 4, 4513. [Google Scholar] [CrossRef] [PubMed]
- Yen, S.T.; Zhang, M.; Deng, J.M.; Usman, S.J.; Smith, C.N.; Parker-Thornburg, J.; Swinton, P.G.; Martin, J.F.; Behringer, R.R. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev. Biol. 2014, 393, 3–9. [Google Scholar] [CrossRef] [PubMed]
- Ohtsuka, M.; Sato, M.; Miura, H.; Takabayashi, S.; Matsuyama, M.; Koyano, T.; Arifin, N.; Nakamura, S.; Wada, K.; Gurumurthy, C.B. i-GONAD: A robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol. 2018, 19, 25. [Google Scholar] [CrossRef] [PubMed]
- Hogan, B.; Beddington, R.; Constantini, F.; Lacy, E. (Eds.) Manipulating the Mouse Embryo, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 1994. [Google Scholar]
- Sato, M.; Akasaka, E.; Saitoh, I.; Ohtsuka, M.; Watanabe, S. In vivo gene transfer in mouse preimplantation embryos after intraoviductal injection of plasmid DNA and subsequent in vivo electroporation. Syst. Biol. Reprod. Med. 2012, 58, 278–287. [Google Scholar] [CrossRef] [PubMed]
- Sato, M. Intraoviductal introduction of plasmid DNA and subsequent electroporation for efficient in vivo gene transfer to murine oviductal epithelium. Mol. Reprod. Dev. 2005, 71, 321–330. [Google Scholar] [CrossRef] [PubMed]
- Takahashi, G.; Gurumurthy, C.B.; Wada, K.; Miura, H.; Sato, M.; Ohtsuka, M. GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system: A novel microinjection independent genome engineering method in mice. Sci. Rep. 2015, 5, 11406. [Google Scholar] [CrossRef] [PubMed]
- Kaneko, T.; Tanaka, S. Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts. Biochem. Biophys. Res. Commun. 2020, 527, 1039–1042. [Google Scholar] [CrossRef] [PubMed]
- Gurumurthy, C.B.; Sato, M.; Nakamura, A.; Inui, M.; Kawano, N.; Islam, M.; Ogiwara, S.; Takabayashi, S.; Matsuyama, M.; Nakagawa, S.; et al. Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. Nat. Protoc. 2019, 14, 2452–2482. [Google Scholar] [CrossRef] [PubMed]
Target Locus | Treatment | No. Mice Treated | No. Pregnant Mice | No. Pups (A) | No. Indels (B) | % (B/A) |
---|---|---|---|---|---|---|
Gene A | 0 min with RNP + dye | 7 | 3 | 13 | 3 | 23 |
3 min with RNP + HA + dye | 6 | 3 | 13 | 6 | 46 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Takabayashi, S.; Iijima, K.; Tsujimura, M.; Aoshima, T.; Takagi, H.; Aoto, K.; Sato, M. Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein. Int. J. Mol. Sci. 2022, 23, 10678. https://doi.org/10.3390/ijms231810678
Takabayashi S, Iijima K, Tsujimura M, Aoshima T, Takagi H, Aoto K, Sato M. Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein. International Journal of Molecular Sciences. 2022; 23(18):10678. https://doi.org/10.3390/ijms231810678
Chicago/Turabian StyleTakabayashi, Shuji, Kenta Iijima, Masumi Tsujimura, Takuya Aoshima, Hisayoshi Takagi, Kazushi Aoto, and Masahiro Sato. 2022. "Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein" International Journal of Molecular Sciences 23, no. 18: 10678. https://doi.org/10.3390/ijms231810678
APA StyleTakabayashi, S., Iijima, K., Tsujimura, M., Aoshima, T., Takagi, H., Aoto, K., & Sato, M. (2022). Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein. International Journal of Molecular Sciences, 23(18), 10678. https://doi.org/10.3390/ijms231810678